Analytic detection of biomolecule analytes (e.g., proteins, nucleic acids, carbohydrates, lipids, etc.) is fundamental to molecular biology. In many applications, it is desirable to detect the presence of one or more particular molecules in a sample. For example, identification of a particular DNA sequence within a mixture of restriction fragments is used to determine the presence, position, and number of copies of a gene in a genome. It is also an integral technique in DNA typing. Analytic detection is also used, e.g., in disease diagnosis and drug development, to determine the presence of a particular antibody or protein, e.g., in a blood sample or large chemical library. Detection of biomolecule analytes is therefore of fundamental value in diagnostic medicine. To meet these needs, many techniques, e.g., DNA blotting, RNA blotting, protein blotting, and immune assays such as but not limited to, ELISA assays, have been developed to detect the presence of a particular molecules or fragment in the midst of a complex sample containing similar molecules.
Western blotting is one tool to identify and quantify a specific protein in a complex mixture. This technique enables indirect detection of protein samples immobilized on a membrane, such as a nitrocellulose membrane, a nylon membrane, or a polyvinylidene fluoride membrane. In a conventional Western blot, protein samples are first resolved by SDS-PAGE and then electrophorectically transferred to the membrane. Following a blocking step, the membrane is probed with a primary antibody (poly- or monoclonal) that was raised against the antigen to be detected. After a subsequent washing step, the membrane is incubated with an enzyme-conjugated secondary antibody that is reactive toward the primary antibody. The activity of the enzyme, such as alkaline phosphatase or horseradish peroxidase, is necessary for signal generation, when the secondary antibody interacts with the primary antibody. Finally, the membrane is washed again, and incubated with an appropriate enzyme substrate (e.g., a chemiluminescent substrate) to produce a recordable signal.
Western blotting substrates are often luminol-based and produce a chemiluminescent signal. Chemiluminescence is a chemical reaction that produces energy released in the form of light. In the presence of horseradish perioxidase, luminol forms an excited state product that emits light as it decays to the ground state. Light emission occurs only during the enzyme-substrate reaction and, therefore, once the substrate in proximity to the enzyme is exhausted, signal output ends.
Because a Western blot is composed of a series of linked techniques that require skill to perform, failure to capture a signal can be caused by many factors. For example, the final step for obtaining the chemiluminescent signal may be time-consuming and subject to human error. This final step typically requires (1) preparing the chemiluminescent substrate solution (e.g., combining a luminol substrate solution with a stable peroxide buffer), (2) placing the antibody conjugate-probed membrane in the substrate solution, (3) incubating the antibody conjugate-probed membrane in the substrate solution (e.g., for at least 5 minutes), (4) preparing a blot membrane or sheet protector, (5) placing the incubated membrane in the blot membrane or sheet protector, and (6) transferring the incubated membrane to an image capturing device. There is a need in the art to replace this process with more efficient and accurate processes. The present disclosure is directed to solving this and other needs in the art.